This invention relates to methods for potentiating the activity of insulin-like growth factor. This invention also relates to a human insulin-like growth factors I and II (IGF-I and II) binding protein (stimulatory form) which has this activity and to methods for producing this binding protein. The binding protein (stimulatory form) has utility in potentiating follicular development in vivo and in vitro, potentiating wound and burn healing, ulcer healing, and for re-epithelialization of damaged tissue of the kidney, lung or skin. The protein may also have utility in potentiating healing of damaged neurons and oligodendrocytes.
In addition, this invention relates to a human insulin-like growth factor binding protein (inhibitory form). The IGF binding protein (inhibitory form) may be used to inhibit tumor growth, inhibit the progression or growth of atherosclerotic plaques, inhibit diabetic retinopathy, and inhibit pulmonary fibrosis.
Human insulin-like growth factor i (IGF-I), also termed somatomedin-C, is a growth hormone dependent growth factor that circulates in blood and is synthesized in many tissues. Growth factor IGF-I is useful as an additive to cell culture media to facilitate in vitro cell growth. Like other growth factors that circulate in plasma, such as EGF, the insulin-like growth factors are bound to a binding protein that forms a carrier protein-IGF-I complex and is believed to serve a transport function. Partially purified forms of this binding protein previously have been shown to inhibit the insulin-like actions of IGF-I. An acid stable binding subunit of this complex has been purified from blood, and its secretion can be stimulated by growth hormone.
Human insulin-like growth factor II is weakly or not at all growth hormone dependent and is synthesized in many tissues. Like IGF-I, it circulates bound to a binding protein that is believed to serve a transport function.
In contrast to the above growth factor binding proteins, extracellular fluids such as spinal, lymph, and amniotic fluids, and tissue extracts of brain, placenta, and pituitary contain forms of IGF binding protein that have different molecular weights and are not growth hormone dependent. They bind both IGF-I and IGF-II with affinities in the 10.sup.10 to 10.sup.9 M.sup.-1 range. An IGF binding protein has also been shown to be secreted by some cell types in culture including human fibroblasts and MDA-231 cells. Impure preparations of the protein from amniotic fluid and a similar protein that is secreted by rat liver cells, have been shown to inhibit the effects of IGF-I and IGF-II on fibroblast DNA synthesis and on sulfate incorporation into cartilage.
However, all of the prior human proteins were actually mixtures of more than one protein. For the first time, the present inventors have developed a method to separate these binding proteins. It has been found that the protein mixture from amniotic fluid actually contained at least two protein constituents. These protein constituents were found to have greatly different activity, one being capable of stimulating or enhancing the activity of IGF-I and IGF-II and one capable of inhibiting the growth factor activity of IGF-I and IGF-II. These two constitutent proteins, whose isolation to substantial purity is described herein for the first time, are referred to for the purposes of this application as IGF binding protein (stimulatory form) and IGF binding protein (inhibitory form), respectively. Experiments have indicated that the proteins may differ in the type of post-translational modifications, possibly disulfide bond arrangements. Such changes may lead to differences in degree of aggregation that may account for the observed differences in cell or matrix binding of the IGFBP and its biological activity.
In addition to the above amniotic fluid IGF binding proteins, an IGF binding protein has been isolated by the present inventors from human fibroblast conditioned media. This form of IGF binding protein potentiates the action of IGF like the stimulatory form from amniotic fluid. It cross-reacts with polyclonal antibodies to the IGF (stimulatory form) but may not be the same protein.
The purified IGF binding proteins (stimulatory form) have an estimated molecular weight between 32-38K daltons. These purified proteins, when adherent to cell surfaces, cause an increase in the amount of IGF that binds to the IGF receptor and to cell surfaces. Surprisingly, this increased amount of bound IGF results in a synergistic potentiation of the IGF activity.
The substantially purified IGF binding protein (inhibitory form) also has a molecular weight of 32-38 KD but does not adhere to cell surfaces and elutes later on DEAE-cellulose columns.
In the discussion which follows, reference to IGF-I binding protein or to IGF-II binding protein should be considered to refer to either or both species, unless the context indicates otherwise. Also, the abbreviation IGFBP may be used to refer to an IGF binding protein.